Hydrolysis of ribonucleic acid containing material

ABSTRACT

DOG FOOD COMPOSITIONS WHICH EXHIBIT ENHANCES FLAVOUR AND ACCEPTABILITY WHEN FED TO DOGS CONTAIN SMALL AMOUNTS, ON THE ORDER OF 4 PARTS PER MILLION AND UP, OF A RIBONUCLESOIDE, SUCH AS ADENOSINE, A 2&#39;&#39;,3&#39;&#39;-RIBONUCLEOTIDE, A SUCH AS 2,3&#39;&#39;-URIDYLIC ACID, A POLY-2&#39;&#39;,3&#39;&#39;-RIBONUCLEOTIDE, A CHEMICALLY HYDROLYZED RIBONUCLEIC ACID-CONTAINING MATERIAL AND/OR CERTAIN OTHER STRUCTURALLY RELATED COMPOUNDS SUCH AS URIC ACID ,URACIL, PYRIMIDINE AND PURINE, AND SALTS AND MIXTURES OF SUCH MATERIALS AND THE LIKE. ENHANCEMENT OF THE FLAVOUR AND ACCEPTABILITY OF SUCH DOG FOOD COMPOSITIONS MAY BE FURTHER IMPROVED BY INCUDING A GLUTAMIC ACID SALT, SUCH AS MONOSODIUM GLUTAMATE. THE COMPOSITIONS MAY BE STABILIZED AGAINST DETERIORATIONS OF THE ENBILIZNG AGENT SUCH AS SODIUM TRIPOLYPHOSPHATE OR A SALT BILIZING AGENT SUCH AS SODIUM TRIPOLYPHOSPHATE OR A SALT OF ETHYLENEDIAMINETETRAACETIC ACID. THE FLAVOR ENHANCING MATERIALS MAY BE ADDED TO CONVENTIONAL DOG RATIONS IN AN AQUEOUS MEDIUM AT THE TIME OF FEEDING OR MAY BE SPRAYED ONTO OR OTHERWISE ADDED TO DOG RATIONS DURING PROCESSING THEREOF. RIBONUCLEIC ACID-CONTAINING MATERIALS, SUCH AS YEASTT MATERIALS, MAY BE SUBJECTED TO CHEMICAL HYDROLYSIS AT A PH ABOVE 12 AND A TEMPERATURE OF 25-55* C. FOR PERIODS OF THREE OR MORE HOURS TO PRODUCE HYDROLYZATE MATERIALS USEFUL FOR ENHANCING THE FLAVOUR AND ACCEPTABILITY OF DOG FOODS.

United States Patent US. Cl. 260-211.5 R I 10 Claims ABSTRACT OF THE-DISCLOSURE Dog food compositions which exhibit enhanced flavor andacceptability when fed to dogs contain small amounts, on the order of 4parts per million and up, of a ribonucleoside, such as adenosine, a2,3'-ribonucleotide such such" as 2',3-uridylic acid, apoly-2',3'-ribonucleotide, a chemically hydrolyzed ribonucleicacid-containing material and/or certain other structprally relatedcompounds such as uric acid, uracil, pyrimidine and purine, and saltsand mixtures of such materials and the like. Enhancement of the flavorand acceptability of such dog food compositions may be furtherimproved'by including a glutamic acid salt, such as monosodiuinglutamate. The compositions may be stabilized againstdeteriorations ofthe enhanced flavor and acceptability by the addition of a stabilizingagent such as'sodium tripolyphosphate or a salt of.ethylenediaminetetraacetic acid. The flavor enhancing materials may beadded to conventional dog rations in an aqueous medium at the-time offeeding or may be sprayed onto or otherwise added to dog rations duringprocessing thereof. Ribonucleic acid-containing materials, such as yeastmaterials, may be subjected to chemical hydrolysis at a pH above 12 anda temperature of 25-55 C. for periods of three or more hours to producehydrolyzate materials useful for enhancing the flavor and acceptabilityof dog foods.

CROSS-REFERENCE TO RELATED APPLICATION This application is a division ofapplication Ser. No. 715,149, filed Mar. 22, 1968;n'ow abandoned.

BACKGROUNI) OF THE INVENTION tides are useful for flavor enhancement ofhuman foods.

Thus, 5-nucleotides such as S' -guanyIic acid, 5-inosinic acid and theirsalts are potent flavor enhancers when incorporated into foods forhumans. Certain 5'-nucleotides,

3,830,798 Patented Aug. 20, 1974 drolysis of ribonucleic acid materialshas been avoided since the hydrolyzates or degradative products contain2- and 3-nucleotides but not 5'-nucleotides.

Moreover, contrary to the effect observed in humans, it has been foundthat the addition of 5'-nucleotides to dog foods produces little or noenhancement of the flavor or acceptability of such foods when fed todogs. There has remained, therefore, an unfulfilled need for materialsWhich will impart a significantly enhanced flavor to dog foods and whichcan be employed on an economical basis in a manner comparable to the useof 5-nucleotides in human foods.

SUMMARY OF THE INVENTION Therefore, among the several objects of thepresent invention may be noted the provision of novel dog foodcompositions possessing improved flavor and acceptability; the provisionof such dog food compositions which may be economically produced andwhich give consistently favorable results when fed to various species ofdogs; the provision of methods of imparting significantly increasedacceptance and palatability to dog foods by incorporating therein smallamounts of certain flavor-enhancing materials; and the provision ofnovel methods of producing certain useful flavor-enhancing materialsfrom readily available ribonucleic acid-containing materials. Otherobjects will be in part apparent and in part pointed out hereinafter.

The present invention is thus directed to an improved dog foodcomposition possessing enhanced flavor and acceptability which comprisesa dog ration and a flavor-enhancing agent from the group made up ofribonucleosides, 2,3 ribonucleotides, poly-2',3'-ribonucleotides,purine, pyrimidine, ribonucleic acid, chemically hydrolyzed ribonucleicacid-containing materials, thymidine, xanthosine, hypoxanthine,xanthine, uric acid, cytosine, S-methyl cytosine, uracil, thymine,orotic acid, adenine, guanine, ureidosuccinic acid, dihydro-DL-oroticacid, dihydrouracil, dihydrothymine, dihydro-6-methyl uracil, allantoinand salts and mixtures thereof. The invention further includes themethod of imparting enhanced flavor and acceptability to dog foods byadding to a dog ration one or more of the above-designated materials.The invention also includes the novel method of chemically hydrolyzing aribonucleic acid-containing material to produce a relativelybacteria-free material useful for imparting enhanced flavor andacceptablility to dog foods.

such-as sodium 5-inosinate and sodium 5'-guanylate, in i combinationwith monosodiumglutamate, have also been found to enhance the magnitude"of the chorda tympani response in rats to stimulation of the tongue.

It is, however, generally recognized by the art that 2'- angly-nucleotides, although isomers of the 5'-nucleotides, possess little orno flavor-enhancing properties. For example, Pat. No. 3,104,171, datedSept, 17, 1963, states that purine and pyrimidine bases, theirnucleosides, and their 2- and 3'-nucleotides have little flavor while5'-nucleotides have very agreeable good taste. A similar statementisfound (page 1, col. 1, lines 44-50) in Pat. No. 3,355,-'

DESCRIPTION OF THE PREFERRED EMBODIMENTS In accordance with the presentinvention, it has been unexpectedly found that 2',3-nucleotides, poly2,3'- nucleotides, nucleosides, and certain other materials areeffective to impart a significantly enhanced flavor or palatability todog foods. In view of the experience of the prior art with suchmaterials as discussed above, this finding is surprising and isapparently attributable to a biological specificity of such materials indogs which differs from that observed in humans as regards the propertyof flavor enhancement. In any event, while the underlying mechanism isnot fully understood, it has been demonstrated on the basis ofactual'test results involving the addition of such materials to dogrations fed to different species of dogs that a positive and definitepreference for such rations over control rations is shown by the variousgroups of dogs tested. It-will be understood that the-expression2',3'-ribonucleotides as employed herein includes 2'-ribonucleotides,-ribonucleotides, 2,3 ribonucleotide's and salts and mixtures thereofand that the expression-poly 2',3- ribonucleotides as employed hereinincludes polyribonucleotides in which the polymer chain terminates in a2'- ribonucleotide, 3-ribonucleotide or 2',3'-ribonucleotide unit, andsalts and mixtures thereof. Exemplary of the 2, 3'-rib0nucleotides whichmay be used in the practice of the invention are purine-based materials,such as 3'-aden ylrc acid, 3'-guanylic acid, 2',3' adenylic acid, 2,3'inosrmc acid, 2' xanthylic acid and pyrimidine-based materlals such as2',3' cytidylic acid and 2',3 uridylic acid. Other useful2,3-ribonucleotide compounds having a free phosphate or a phosphate saltradical in the 2'- 3'- or 2' and 3' positions of the ribose portion ofthe molecule are known to the art. Similarly useful in the presentinvention are poly 2,3' ribonucleotides which are polymers in whichunits of ribonucleotide molecules are joined together to form polymersof various chain lengths and which, as aforesaid, have a2'-ribonucleotide, 3'-ribonucleotide or 2',3'-ribonucleotide unit at aterminal portion of the polymer chain. Since the separation of2'-ribonucleotides from 3'-ribonucleotides and of 2'- and3-ribonucleotides from 2',3'-ribonucleotides and the separation of poly2,3' ribonucleotides from each other is diflicult, it is generally moreconvenient to use an admixture of such materials in pure or impure formin the practice of the invention. As described hereinafter, mixtures of2'-, 3'- and 2,3'-ribonucleotides can be prepared in accordance with theinvention by the chemical hydrolysis of ribonucleic acid-containingmaterials such as yeasts and the resulting hydrolyzates containing suchmixtures can be directly utilized in carrying out the practice of theinvention.

The useful ribonucleotides include adenosine, guanosine, cytidine anduridine or other purine or pyrimidine bases combined with ribose.

In addition, various other compounds have been found useful for flavorenhancement of dog foods in accordance with the invention. These includepurine, pyrimidine, ribonucleic acid, thymidine, xanthosine,hypoxanthine, xanthine, uric acid, cytosine, S-methyl cytosine, uracil,thymine, orotic acid, adenine, quanine ureidosuccinic acid,dihydro-DL-orotic acid, dihydrouracil, dihydrothymine, dihydro-6-methyluracil, and allantoin.

Where salts of any of the foregoing compounds or materials are employed,it is generally preferred to use alkali metal salts such as sodium orpotassium salts or alkaline earth salts such as calcium salts. Theselection of the particular salt or salts used may in part be governedby the composition of the dog ration to which the flavorenhancingcompounds or materials of the invention are added and by thenutrititional requirements of the dogs being fed.

A practical and relatively inexpensive mode of preparing usefulflavor-enhancing materials for employment in the practice of theinvention involves the chemical hydrolysis of ribonucleicacid-containing materials. The expression ribonucleic acid-containingmaterials as used herein designates polymeric compounds or materialsfound in living tissues and including monomeric units consisting ofpurine or pyrimidine bases combined with ribose units. In the case ofnucleotides and polynucleotides, the units include phosphate groups atthe 2',3'- or 2- and 3'-carbon positions whereas the nucleosides arephosphate-free. Useful ribonucleic acid-containing materials includesuch natural sources as yeast (e.g., dried brewers yeast or torulayeast) and animal tissues. As is known in the art, the chemicalhydrolysis of such materials with aqueous alkali produces a hydrolyzatecontaining a mixture of 2- 3- and/or 2,3' ribonucleotides, and/or poly2',3' ribonucleotides and/ or ribonucleosides, the exact composition ofthe hydrolyzate being dependent upon the extent of hydrolysis (i.e.,partial or complete), the conditions "5r hydrolysis and the nature ofthe starting material. In 'the prior art, chemical hydrolysis ofribonucleic acid-containing materials has generally beencarried out at apH below 12, for example, by employing 1.5-2.0 milliequivalent ofalkaline material per gram of yeast starting material. The

hydrolysis has also been carried out at room or elevated temperaturesand for a sufiic ient length of time to give the desired degree ofhydrolysis.

The prior art hydrolysis procedures may be utilized to produce useablehydrolyzates for accomplishing the purposesof the present invention.However, the conditions of alkaline hydrolysis previouslyemployed havebeen found to contribute to an objectionablebuildup of certainthermophilic bacteria. In accordance with themvention, we have foundthat a relatively bacteria-free hy drolyzate can be produced by carryingout the hydrolysis at a pH of at least 12 and 'maintaining'thehydrolysismixture at a pH in excess of 12. Since the pH normallydecreases slightly (0.2-0.4) during .the course of hydrolysis, it ispreferred to operate at a pH in excess'of 12.5 or 13. For example, usingyeast as the ribonucleic acid-containing material, approximately 3.5milliequivalent of an alkaline material, such as potassium hydroxide,per gram of yeast material has been found to produce 'the desired pH.With respect to temperature, the hydrolysis may be carried out at roomtemperatures (i.e., 15. C.- 25 C.) or at elevated temperatures.Preferably, the hydrolysis is carried out at temperatures betweenapproximately 25 C. and 55 C. The hydrolysis should be continued for atleast three hours and, for the purposes of the present invention, ispreferably conducted, for a period of 12 to 24 hours. It will beunderstood that various alkaline materials known to the art, such assodium hydroxide and potassium hydroxide, may be employed.

The hydrolyzates resulting from the above hydrolysis procedures are inthe form of aqueous slurries or suspensions and may be used directly inthe practice of the invention by being sprayed onto dog rations orotherwise added thereto. If desired, the hydrolyzates may be neutralizedby the addition of an acid, such as acetic'acid, phosphoric acid orhydrochloric acid, immediately, prior to being applied to dog rations. 1A

An important feature of the present invention is-our finding that theaddition of only small amounts of the flavor-enhancing agents of theinvention is suflicient to impart a significant flavor-enhancingeflFect. Thus, in general, as little as 4 parts per million, based uponthe dog ration, will impart a definite enhancement in flavor andacceptability. Preferably, between approximately 8 and parts per millionof the flavor-enhancing compound or material is added to the dog rationto obtain the desired flavor-enhancing effect. In using a hydrolyzateprepared as described above as the flavor-enhancing agent, we may employbetween 0.2 and 2 grams -(dry weight) per pound of dog ration,preferably 1 gram per pound of dog ration, to secure the desired degreeof flavor enhancement. This is equivalent to approximately 80-400 partsper million parts of dog ration of the active ribonucleoside, 2','3-ribonucleotide, polyribonucleotide and poly-2',3'-ribonucleotideflavor-enhancing materials contained in thehy drolyzate. It will 'beunderstood that the useful hydroly zates described above contain amixture'of such polymeric and monomeric materials and salts thereofwhich, in admixture, produce a distinct flavor-enhancing effect.

The flavor, acceptability and/or palatabilit'y of the dog foodcompositions of the invention may be further enhanced by adding aglutamic acid salt to a dog ration in combination with one or moreof theaforementioned flavor-enhancing agents- Mono'sodiurn glutamate isfthepreferred salt for use, but it will be understood thatother salts suchas the potassium and calcium salts may also be employed. In general,atleast 20 parts'per million parts of dog ration of such a glutamicacidsalt should be used to a further enhancement or, flavor.

It has further been found,in accordance,withthe-dhvention that ournovehdog foodcompositions may be stabilized against deterioration oftheflavor-enhancing properties during storage of the compositions forextended periods of time by adding thereto small eamountsof-astabilizing agent which funetons to stabilize the phosphate bond in theribonucleotide molecules or to stabilize the molecule as a whole or anypart thereof. Among the stabilizing agents which may be used are sodiumtripolyphosphate, orthophosphoric acid and salts ofethylenediaminetetraacetic acid.

' The flavor-enhancing agents of theinvention or mixtures thereof may beadded to conventional dog rations, such as that sold under the tradedesignation Purina Dog Chow for example, by any means desired eitherduring processing of the dog food or before or at the time of feedingthe dog food. As an illustration, the dog food compositions of theinvention may be conveniently formed by spraying the flavor-enhancingagent, carried in an aqueous medium, onto the dog ration or food duringprocessing and prior to packaging thereof. Upon evaporation of theaqueous carrier, the agent is carried in a dry form by the dog rationparticles and is distributed therethrough. The flavor-enhancing agentsmay also be added to the dog ration or food in the water used to moistenthe food at the time of feeding. Further, the agents may be mixed with adry carrier and dusted onto the the dog ration or food.

It will be understood that the flavor-enhancing materials of theinvention may be added to conventional dog rations or foods of varioustypes (e.g., dry and moist dog foods) and may be added alone or incombination with other foods, ingredients or additives to accomplish theobjectives of the invention. a

The following examples illustrate the invention.

In the experimental work described in the following examples, unlessotherwise stated, the dog ration employed was that sold under the tradedesignation Purina Dog Chow" by Ralston Purina Company. This productcontains as ingredients meat and bone meal, wheat germ and cobaltcarbonate. By analysis, the product contains not less than 23% crudeprotein and 8% crude fat, and not more than 4.5% crude fiber and 10%ash.

The dogs employed in the experimental work included poodles, redbonecoonhounds, English setters, Labrador retrievers, springer spaniels,beagles, pointers and greyhounds. The dogs were randomized by groups,each containing 10 dogs, and there were 5 test dogs in each pen duringthe studies. The dogs were fed on an individual basis with the rationpans being rotated so that the same ration was not offered on the sameside of the pen each day. The ration consumption of each dog wascalculated and recorded for the test period indicated in the results setout below.

The statistical significance of the test results was determined by usingthe Wilcoxons matched-pairs, signedranks test (American StatisticalAssociation Journal, September 1965, pages 866867).

In the tabulated results given in the tables below, the first figureunder each ration column represents the total number of pounds of theration which the group of dogs consumed, the dogs having equal access toboth this ration and a second test or control ration, and the secondfigure (given in parentheses) represents the number of dogs whichconsumed more of the one ration in preference to the second ration.Where the second figure includes the fraction /2, this means that atleast one dog showed equal preference for two rations.

EXAMPLE 1 The six materials set forth below were added in water toPurina Dog Chow at the time of feeding to determine whether thematerials at the levels tested increase acceptability of the ration tothe test dogs. The results are given in Table 1.

TABLE 1 Ration number Amount of test material A (control), B, 8 C, 80Signifi- (ied wet-alr-dry basis) 0 p.p.m. p.p.m. p.p.m. canoe Test 1(guanine):

Phase 1=15 dogs, 5 days 31. 6(3) 46. 102) P .01 Phase 2=15 dogs, 5 days22. 0(0) 53. 1(15) P .0l Phase 3=15 dogs, 5 days 36. 6(6y 37. 06%) N.S.Test 2 (guanoslne):

Phase 4=15- dogs, 5 days..- 25. 8036) 47.30am P .01 Phase 5=15 dogs, 5days... 30. 2(23/ 46 401%) P .01 Phase 6=15-dogs, 5 days-.- 27.6(4) 1(11P .01 Tistdii t(g)uanosine-3-(2) phosphoric acid-sodium salt,

y ra e Phase 7=15 dogs, 5 days 20. 1 (2) 41. 7(13) P 01 Phase 8=15 dogs,5 days 22. 0(2) 41. 303) P .0l Phase 9 =15 dogs, 5 days 26. 2(4) 38.8(11) P .01 Test 4 (lnosine);

Phase 10=l5 dogs, 5 days 28.1(3) 42. 2(12) P .03 Phase 11=15 dogs, 5days 25. 6(4) 39. 701) P .02 Phase 12 15 dogs, 5 days 28. 1(236)41.3(12}) P .01 Test 5 (cytidine):

Phase l3=15 dogs, 5 days 32. 1(4) 40.7(11) N.S. Phase 14=15.d0gs, 5 days20.9(5) 49 204%) P .01 Phase 15=15 dogs, 5 days 3013(6) 1 9 N.s. Test 6(midine):

Phase 16=15 dogs, 5 days 27. 2(4) 2(11) P .01 Phase 17=15 dogs, 5 days20. 9(2) 44. 8(13) P .01 Phase 18=15 dogs, 5 days 23. 7(4) 43. 5(9) N.S.

The results show that each of the six materials increased acceptabilityat both the 8 and p.p.m. levels as compared with the control ration.

EXAMPLE 2 Example I was repeated with six additional materials as testmaterials using six pens of rotated dogs. The results are given in Table2.

- Ration Amount test material B, 8? 2 (fed wet-aig-dry basis) 1 1111411.

Test 1 (guanine): 7 7

Phase 1:15 dogs, 5 days I 53. 6(13) I Phase 2=15 dogs, 5 days 0) Phase3=15dogs,'5 days 158.2(5) K Test 2 (guanosine): v

Phase 4=15 dogs, 5 days 32.'4(2%) 57. 002%) ...e 1; P .00l Phase 5=15dogs, 5 days... 33.3(2) 60.7(13) -P .QQ5 Phase 6=15 dogs, 5 days 30.4(4)51. 0(11) P .02 Test 3 (gusnosme-3-(2) phosphoric acid, sodium salt, I

hydrate): I 5

Phase 7=15 dogs, 5 days 36 8(4) 50. 3(11) P .04

Phase 8=15 dogs, 5 days 31. 3(3) 59.0(12) P L005 Phase 9=15 dogs, 5days- 48. 3(10) 45. 1(5) N.S..

Test 4 (inosine): I

Phase 10=15 dogs, 5 days P .03r

Phase 11=15 dogs, 5 days... 66.4(13k) P .005 v Phase 12=15 dogs, 5days... 43. 5(5) 52. 5(10) N.S.

Testg)(mixed isomers of 2-cytidy1ic acid and 3-eytidylic V 801 I 1 Phase13=15 dogs, 5 days 35. 2(4) 53. 3(11) P .05 Phase 14:15 dogs, 5days...-. 30.3(2) 5- 63. 903) P .005 5 Phase 15=15 dogs, 5 days 42. 60%)51.00%) N.S. Test 6 (2,3-uridylic acid):

Phase 16:15 dogs, 5 days 31.9(3) 49. 6(12) P .05

Phase 17=15 dogs, 5 days..-.. 27. 00%) 59. 503%) P .001,

Phase 18:15 dogs, 5 days 44. 5(4) 53. 1(11) NS.

The results show that the six materials efiectively in- I creasedacceptabillty of the test ratlons compared With Examples 1 and 2 wererepeated,usingguanosineflawne the control ratron. and a mixture ofguanosine arrdmonosodiurn 'lutarnate 1 fth 11 tthothtth cog The resu is0 0 Owlng es W a e m as the test matenals. The guanosmeused was obtainedbinfltion 0f guanosme and monosodlum glutamate P from two differentsources of snpply.The results-'ar "given duced greater acceptabilitythan guanosine alone. in Table 3,

TABLE 3 Ration number 1 2 3 4 5 6 7 Ration, Purina Dog Chow containing:

Guanosine, p.p.rn 0 4 8 0 4 8 0 Monosodium gluta p.p.m 0

Feed consumption-fed wet (air-dry basis) No. No. Phase dogs days 15 5511.4(5) 59.6(10) 15 5 40. MW) 45. y) 15 5 52.4(234) 61.10% a 15 540.7(6)

Ration number 8 9 10 11 12 Significance Ration, "Purina Dog Chow"containing: 7

Guanosine, p.p. m 4 8 q 4 8 Monosodium glutamate,

p.p.m 20 '40 0 20 40 Feed consumption-fed wet (air-dry bBSlS) No. N 0.dogs days 9 EXAMPLE 4 .fl'heeifect of adding iribon ucleic acid andhydrolyzed ribonucleic acid, respectively, in solution to Purina DogChow at the time of feeding to groups of test dogs was determined, Theresults are given in Table 4.

10 group consisted of 10 dogs and the test period or phase in eachinstance was 5 days. The results are given in Table 6.

TABLE 4 Signifi- Ratlonnumber 1 (control) 2 3 ounce Percent Purina DogChow" 100 100 100 RNA, p:p.m. 8 Hydrolyzed RNA, n n m 8 Food consumptionted wet (airdry basis):

- Phase 1 =30dogs,5-days.- 49.8() 103.7(30) P .001 Phase 2=30 dogs, days55. 16%) 89. 004%) P .001 Phase 3=0 dogs, 5 days. 65. 5 87. 0(20) P .03

TABLE 0 Food consumption of- Test v v 20 material The results show thatboth materials were effective in com-o1 -1 33 increasing acceptability.a "Purina Signifi- 7 Test material Dog Chow Dog Chow" cance EXAMPLE 5Purine"... 24.5(2 42.20% P .10 I was as; are; 2322 1 ne- 'll' 1e effecton the acceptability of dog IZtlOIlS of Cyudmm" 153(0) 3mm) P m5guanosme anda combination of guanyhc acid, undyhc Thymi 'negggta) 42.1w)P .o aci ,i9y d'y ic acid. an yl c d added in solution Ema-, 1 a,; 1}?28 to,'Pur1na Dog Chow at the time of feeding was deex-ag m phosphateoiuridyllc 14.1 m 33.1 9 P .01 rn 9 =1 Te$l11taretfrth in Table Hgporanthine 23.20% 41.20% P .01 4 r Xanthine. 215(2) 39.7(8) P .10

Um acid--- 1017(0 26.2(10) P .005 Cytoslne-. 18. 5(1) 28.9(9) P .005S-methylcytos 19.4(1) 36.8(9) P .01 Uracil 18 2(1) 43.3(9) 1 .01

, TABLE 5 Signifi- Ratlon number 1 (control) 2 3 canoe Percent PurinaDog Chow 100 100 100 Guanostne..... 8 2,3' g'uanyll'c acid, p.p;m'-. 22',2.'-uridyllc acid, p.p.m 2 2",3-cytldylie acid, n p m 22',3-a'rienylic acid, p.p.m .Y 2 Food consumption-ted wet (air-drybasis):

Phase l=15 dogs, 5 days 30. 7(2) 903) P .005 Phase 2=15 dogs, 5 days...26. 7(3) 61.1(12) P .005 Phase 3=15 dogs, 5 days 25. 4(2) 50. 303 P .00l

The results show that each of the materials tested improvedacceptability of the ratiomwhen added at a level @The results ,show--that the combination of the four named materials improvedacceptability to a greater degree than guanosine alone.

l V v E fi effect of various materials on the acceptability of dog''rjations was determined. The materials were added in water ,to the dogrations at the time of feeding. Each test of 10 ppm.

EXAMPLE 7 The effect of brewers dried yeast and hydrolyzed brewers driedyeast on the acceptability of dog rations was determined. The materialswere added in the water used to moisten the rations at the time offeeding. The results are given in Table 7. The placebo below was soymeal.

TABLE 7 Signifi- Ration number 1 (control) 2 3 cance Purina'Dog Chow,"grams"--- 452. 67 Control (placcbo"), grams... 1. 0 Brewer's driedyeast, grams"- Hydrolyzed Brewer's dried ye Feed consumption-ted wet(air-dry bas Phase 1=30 dogs, 5 days 58.3 P .0001 Phase 2 30 dogs, 5days.. 65. 0(8) 89. 8(2 P .005 Phase 3'=30 dogs, 5 days 47.66%)107.0(2534) P .0001

The results show that both materials increased acceptaimproveacceptability and that the hydrolyzed yeast mability of the action andthat the hydrolyzed yeast material terials render the rationsignificantly :7 mo t b le rendered the ration significantly moreacceptable than did than do the non-hydrolyzed material's thenon-hydrolyzed yeast material.

EXAMPLE 8 The elfect of a period of three months storage ontheacceptability of stabilized and unstabilized brewer's yeast Theeifect of hydrolyzed brewers yeast at two ditferent materials wasdetermined. The materials were sprayed levels on the acceptability ofdog rations was determined. onto the Purina Dog Chow rations prior tofeeding. The The yeast material was sprayed on Purina Dog Chow 10results are given in Table" 10'. TABLE 10 Ration numb r 1 v I PercentPurina Dog Chow 100 J' I I Ugsltabil ized hydrolyzed brewer's yeast,ml./lb. "Dog 7 W. Hydrolyzed brewers yeast stabilized with sodium Vtripolyphosphate and orthophosphon'c acid, mL/lb. "Dog Chow.

Hydrolyzed yeast stabilized with sodium salts or ethyl- 1 gifidiamlne,tetreacetic acid and HCl, mL/lb. Dog

ow. Feed consumption-led wet (air-dry basis):

Phase 1=30 dogs, days 53.9(3) 93.0(27) P .0001 Phase 2=30 dogs, 5 days65. 8(10) 84. 6(10; P .05 Phase 8=30 dogs, 5 days 63. 9(9) 87. 7(21 P.02

before feeding the test dogs. The results are given in Table 8.

TABLE 8 Signifi- Ratlon number e 1 2 3 cance Percent Purina Dog Chow 100100 100 Hydrolyzed brewer's yeast, gramllb 0 0. 2 1. 0 Feedconsumption-fed wet (air-dry bas Phase l= dogs, 5 days 65. 9(3) 116.5(27) P .0001 Phase 2=30 dogs, 5 days" 75. 365%) 110. 2(2636) P .001Phase 3=30 dogs, 5 days 62. 0(4%) 119. 6(25l6) P .0001

The results show that the yeast material significantly The results showthat the unstabilized material is inimproved acceptability of the dogration both at 0.2 gms./ ferior, as regards enhanced acceptability, toboth of the lb. (440 p.p.m.) and at 1 gm./lb. (2,20Q p.p.m. stabilizedmaterials.

EXAMPLE 11 EXAMPLE 9 40 The effect of hydrolyzatron time and temperatureon The eifect of non-hydrolyzed and hydrolyzed brewers the acceptabilityof hydrolyzed brewers yeast materials yeast and torula yeast,respectively, on the acceptability was determined. The varioushydrolyzed" materials were of dog rations was determined. The materialsin the form sprayed onto the Purina Dog ,ChowL rations. prio to ofaqueous media was sprayed onto the Purina Dog feeding. The resultsare'given in Table" 1 I TABLEII Ii Ration number 1 2 3 '4' I H H Percent"PurinaDog Chow 100 00 g Brewer's yeast (1 g./lb. "Dog Chow) hydrolyzedatemp C i 25 25 25 35 No. hours proce 8 15 3 Feed consumption-ted wet(ah-dry basis):

Phase 1=30 dogs, 5 days 90. 3(1116) 102. 2(18M) Phase 2=30 dogs, 5 days80. 7(1 104. 5(20) Phase 3=30 dogs, 5 day 70. 1(5) 113. 4(25) Phase 4=30dogs, 5 days.- f 59. 2(10) Phase 5=30 dogs, 5 days Phase 6=30 dogs, 5days--.. 4(3 ,3 Phase 7=20 dogs, 5 days.-. 0%) (1 .2) Phase 8=20 dogs, 5day Phase 9:20 dogs, 5 days.

Chow ration prior to feeding. The results are set forth Table Theresults show that products resultiggjrom hydrql;

' TABLE 9 Ration number v 1 I 3 Percent oi V 100. 100 100 Control 1(water)- Control 2 (water) Non-hydrolyzed brewers yeast 25 Hydrolyzedbrewer's yeast Non-hydrolyzed torula yeast p I 25 Hydrolyze'd tomlayeast. 25

1 .ooo1 52-323: Phase 4=ao dogs, 5 days- 7 48. 916%) 17. seem 1 '.o1Phase 5=30 dogs, 5 days- 52 .jQ(8) 64.8(22) P .02 Phase e=3o dogs,5daysg 46.8(5) 71.7 25 r .ooo1

The results show that two types of yeast, in both ysis at 35, C. areeffective than those resulting from the non-hydrolyzed and hydrolyzedform, significantly hydrolysis at 25" C."and.that hydrolysis for fifteenhours EXAMPLE 12 Commercial ribonucleic acid g.) was added with mixingto 0.3N potassium hydroxide (200 ml.) until a clear light brown solutionwas obtained. This solution was incubatedat 37 C. for a period ofeighteen hours and then neutralized to a pH of about 8 withconcentratedhydrochloric acid. The final volume was adjusted to 250 ml. with water.Using the same quantity of ribonucleic acid and neutralized'potassiumhydroxide as above (plus a small amount of additional potassiumhydroxide to aid solution), a solution of non-hydrolyzed ribonucleicacid was prepared, as well as a blank containing neutralized potassiumhydroxide only. These preparations were the preparations employed inthetests described in Example 4 with the results given'in Table 4 beingobtained.

EXAMPLE 13 Dry brewer's yeast (700 g.) was slurried with 0.3N sodiumhydroxide (4000 ml.) in a Waring Blender. The solution wasincubated fora period of twenty-two hours at 37" C. and neutralized with concentratedhydrochloric acid. Water was'added to bring the total volume to 5200 ml.A suspension of brewers yeast (700 g.) in neutralized sodium hydroxide(4000 ml.) was prepared and diluted to 5200 ml. to serve as a controland a similar solution containing no yeast was prepared to serve as ablank. These materials were'the materials employed in the testsdescribed in Example 7 with the results given in Table 7 being obtained.

EXAMPLE 14 Each of three portions (600g) of dry brewers yeast wasslurried with water (2000 ml.). To this was added 9. 2151 potassiumhydroxide (98 ml.) and water (900 ml.). After mixing, each suspensionwas incubated at 37 C. for eighteen hours and then neutralized to a pHof about 7 with concentrated hydrochloric acid. To one resultinghydrolyzate was added sodium tripolyphosphate (273 g.) plus enoughphosphoric acid'to bring the pH to about 7. To'='a secondhydrolyzate'was added disodium ethylenediaminetetraacetate' (41 g.).Nothing was added to the third hydrolyzate. Water was added to bring thevolume of each preparation to 4200 ml. These preparations were thepreparations employed. inthe tests described in Example 10 with theresults given in Table 10 being obtained.

EXAMPLE 1s q-Dry brewersyeast (770 g.) was slurried in water with theaidof a blender. .To this was added 9.15N potassium hydroxide(130fml.)and enough water to bring the total volume to 3900 ml. Aftereighteen hours incubation at 37 C., theresulting hydrolyzate wasneutralized with concentrated hydrochloric acid (70 ml.), and diluted to5400 m1. One sample ofv the hydrolyzate (800 ml.) was diluted by theaddition of water (3200 ml.) and marked as supplement A. A second sampleconsisting of 4000 ml. of thehydrolyz'ate solution was marked assupplement B. A third sample consisting of 4000 ml. of water was markedas supplement C. These samples were the ones employed in the testsdescribed in Example 8 with the results given 'in Table 8 beingobtained.

X EXAMPLE 16 ,Example 11 was repeated except that the hydrolysisconditionswere varied as follows:

i Q 'Condltions at Temperature, C.

Time 14 The results of feeding the hydrolyzates produced to dogs showedthat the products resulting from hydrolysis at the higher temperatureand for longer periods of time tended to provide greater acceptability.

EXAMPLE 17 Dry brewer's yeast (17.5 lb.) was added to water (97.5 lb.),and the mixture slurried to an even consistency. To this was addedpotassium hydroxide (3.95 lb.) dissolved in water (4.0 1b.). Thisproduced a concentration of 3.5 meq. potassium hydroxide per gram ofyeast. After a period of eighteen to forty-two hours at roomtemperature, portions of the slurry were removed, neutralized withconcentrated hydrochloric acid and sprayed onto Purina Dog Chow. Uponbeing fed to dogs, it was found that rations containing the thusproduced hydrolyzate produced greater acceptability than did controlrations.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods and productswithout departing from the scope of the invention, it is intended thatall matter contained in the above description shall be interpreted asillustrative and not in a limiting'sense.

What is claimed is:

1. The method of chemically hydrolyzing a ribonucleic acid-containingmaterial comprising the steps of mixing, in an aqueous medium, aribonucleic acid-containing material with a sufiicient quantity of analkaline material to produce a pH of at least 12 and maintaining theresulting mixture at said pH and a temperature at least equivalent toroom temperature for a period of at least three hours.

2. The method of chemically hydrolying a ribonucleic acid-containingmaterial as set forth in claim 1 wherein said temperature is betweenapproximately 25 C. and 55 C.

3. The method of chemically hydrolyzing a ribonucleic acid-containingmaterial as set forth in claim 1 wherein said period is betweenapproximately 12 and 24 hours.

4. The method of chemically hydrolyzing a ribonucleic acid-containingmaterial as set forth in claim 1 wherein said pH is approximately -13.

5. The method of chemically hydrolyzing a ribonucleic acid-containingmaterial as set forth in claim 1 wherein the ribonucleic acid-containingmaterial is yeast and the quantity of alkaline material is 3.5milliequivalent of alkaline material per gram of yeast.

6. The method of chemically hydrolyzing a ribonucleic acid-containingmaterial comprising the steps of mixing, in an aqueous medium, aribonucleic acid-containing material with a sufficient quantity of analkaline material to produce a pH of at least 12 and maintaining theresulting mixture at said pH and a temperature at least equivalent toroom temperature for a period of at least three hours therebyproducing ahydrolyzate containing a mixture of materials selected from the groupconsisting of 2'-ribonucleotides, 3'-ribonucle otides, 2',3'-ribo'nucleotides, poly-2,3-ribonucleotides and ribonucleo'sides. I,

7. The method of chemically hydrolyzing a ribonucleic acid-containingmaterial as set forth in claim ti wherein said temperature is betweenapproximately. 25 1C. and

8. The method of chemically hydrolyzing aribonucleic acid-containingmaterial as set forth in claim 6 wherein said period is betweenapproximately. 12 and 24 hours. i 9. The method of chemicallyhydrolyzing a ribonucleic acid-containing material as set forth'in claim6 wherein said pH is approximately 12.5 13.

10. The method of chemically hydrolyzing a ribonucleic acid-containingmaterial as set forth in claim 6 wherein the ribonucleic acid-containingmaterial is yeast 3,338,881 8/1967 Wiley? 2 6()- -2 l1.5 R and thequantity of alkaline material is 3.5 milliequivalent 3,457,254 7/1969Yano 'etf ,jal." 260+- 211 '.5 R of alkaline material per gram of yeast.

v JOHNNIE R. BROWN, PrimaryExaminer References Cited 6 V v UNITED STATESPATENTS 3,382,231 5/1968 Hirahara. et al. 260-2115 R 3,337,530 8/1967Hanze 260-2ll.5 R

